小麦TaGAPC定点突变体基因载体构建及其原核表达

Construction of TaGAPC Site-directed Mutagenesis Gene Vector and Prokaryotic Expression Analysis from Wheat

为了研究催化活性半胱氨酸Cys154、Cys158残基对小麦细胞质甘油醛-3-磷酸脱氢酶(Ta GAPC)蛋白酶活性的影响,利用重叠延伸PCR法分别将这2个位点的半胱氨酸突变成丝氨酸,并分别连入p ET28a(+)原核表达载体。在25℃条件下,Ta GAPC及其定点基因Ta Cys154S/Ta Cys158S经0.25 mmol/L IPTG诱导后高效表达,SDS-PAGE电泳检测结果显示,目标重组蛋白均为可溶性且大小约为40 k Da,与预测结果一致。在此基础上,经超声波破菌及镍柱纯化获得3种纯化重组蛋白。这为后续Ta GAPC及其定点突变体蛋白酶活性测定及活性位点分析提供试验材料。

In order to elucidate the roles of catalytic active Cys154,Cys158 in Triticum aestivum L. cytoplasmic glyceraldehyde-3-phosphate dehydrogenase（ Ta GAPC）,these two cysteins were site-directed mutated into serine by overlap-extension PCR. Then recombinant prokaryotic expression vectors of Ta GAPC and its mutants Ta Cys154 S /Ta Cys158 S were constructed and transformed into E. coli BL21. The recombinant protein were induced by 0. 25 mmol / L IPTG at 25 ℃ and subsequently purified by Ni2 ＋-IDA column. These researches could lay the foundation for the enzyme activity assay of Ta GAPC and its mutants Ta Cys154 S / Ta Cys158 S and cysteine active sites.